Process for the enzymatic extraction and purification of a glycopeptide obtained from animal organs,useful as a drug

ABSTRACT

Glycopeptides are extracted from the gastric mucosa or duodenum of swine by hydrolysis of the animal organ under specified conditions, followed by separation of acidic hydrolysis byproducts and dilution of the extraction product with a nonsolvent to precipitate the glycopeptides.

United States Patent [72] Inventors Gianlranco Bertellini Maslianico; Adriano Butti, Tavernola; Giuseppe Prino, Milan, all of Italy [211 App]. No. 37,886

[22] Filed May 15, I970 [45] Patented Sept. 21, 1971 [73] Assignee Prephar Prospection De Recherches Pharmaceutiques S.A. Sehaffhausen, Switzerland Continuation-impart of application Ser. No. 583,070, Sept. 29, 1966, now abandoned.

[54] PROCESS FOR THE ENZYMATIC EXTRACTION AND PURIFICATION OF A GLYCOPEPTIDE OBTAINED FROM ANIMAL ORGANS, USEFUL AS A DRUG 10 Claims, No Drawings [52] US. Cl 195/29,

[51] Int. Cl C07g 7/026, C12d 5/06 [50] Field of Search 195/29; 260/ 1 I2 [56] References Cited UNITED STATES PATENTS 2,217,264 10/1940 Weizmann l95/29 3,518,243 6/1970 Butti et al. 260/112 FOREIGN PATENTS 1,544,225 l0/l968 France OTHER REFERENCES Glycoproteins, Gottschalk, Dec., 1965, pp. 235- 240, 258- 261, 364- 366, 548- 549 and 558- 559 Primary Examiner-Howard E. Schain Attorney- Flynn & F rishauf ABSTRACT: Glycopeptides are extracted from the gastric mucosa or duodenum of swine by hydrolysis of the animal organ under specified conditions, followed by separation of acidic hydrolysis byproducts and dilution of the extraction product with a nonsolvent to precipitate the glycopeptides.

PROCESS FOR THE ENZYMATIC EXTRACTION AND PURIFICATION OF A GLYCOPEPTIDE OBTAINED FROM ANIMAL ORGANS, USEFUL AS A DRUG RELATED APPLICATION This application is a continuation-in-part of Ser. No. 583,070, filed Sept. 29, 1966 and now abandoned.

FIELD OF INVENTION This invention relates to the extraction and purification of a glycopeptide, from certain animal organs of the digestive ap paratus of swine (gastric mucosa or duodenum), which have proved to be useful as a drug for the cure of inflammatory diseases in general and particularly of ulcers.

BACKGROUND OF INVENTION.

The existence in animal organs of macromolecules of composite nature intermediate between that of proteins and that of polysaccharides has been demonstrated and these substances have been termed glycoproteins. The structure of these macromolecules may, by a rough approximation, be supposed as resulting from the combination of one or more molecules of a polysaccharide with one or more molecules of a protein. This combination is due to covalent linkages and therefore the whole structure is not an adduct or a complex but rather a single molecular species. In some types of glycoproteins the proteic moiety may prevail whereas in others the polysaccharide moiety may prevail. The latter types are therefore usually denominated glycopeptides. To this class belong some substances having a very high biological importances such as, for example, those that characterize the blood groups.

SUMMARY OF INVENTION In accordance with the present invention, there is provided a process for the preparation of a glycopeptide from the gastric mucosa or duodenum of swine, comprising:

a. hydrolyzing the animal organ in water at a temperature of from about 50 C. to about 100 C. for from about to about 45 minutes at a pH of from about 1 to about 10;

b. acidifying the hydrolysis product of (a) whereby a precipitate and a liquid product are formed and separating said precipitate from the liquid product when hydrolysis of (a) is conducted at an alkaline pH, and centrifuging the hydrolysis product of (a) to separate a liquid product from solid matter when hydrolysis of (a) is conducted at an acidic pH;

c. subjecting the product of (b) to enzymatic proteolysis with papain or trypsin; and

d. diluting the product of (c) with a nonsolvent therefor, whereby a glycopeptide is precipitated therefrom.

In accordance with the invention, there is also provided a glycopeptide extracted as described by said process. The glycopeptide comprises, in approximate weight percent:

hcxosamine hexose 27-3l aminoacids l3-l8 neuraminic acid derivatives 4-6 and substantially free of sulfur, phosphorus and uronic acids.

SPECIFIC EMBODIMENTS OF THE INVENTION peratures approaching C., are preferred. The pH value can be adjusted to values which can range from I to 10 by suitable additions of acids or alkalies. Values of pH higher than 10 must be avoided since at such pH values the material is quickly demolished. For the same reasons, at high pH values (above 9) a particularly careful control of temperatures and heating times must be exercised.

At the end of the hydrolysis stage, it may be convenient to effect the precipitation of all of the organic substances having acidic character such as, for example, the sulfur-containing mucopolysaccharides. Hence, if the hydrolysis has been conducted at an alkaline pH, the mixture can be brought again to an acidic pH by adding a weak acid (eg acetic acid) thereby causing the precipitation of these substances. The filtrate is then freed from the proteic drosses by treatment with an enzyme (e.g. papain or trypsin) in a proper pH (from 5 to 7) condition. It can also be convenient to effect, before the enzymatic treatment, a preliminary precipitation of most of the proteic drosses by adding a suitable reactant, such as, for example, trichloroacetic acid. By diluting the filtrates obtained from these treatments with organic solvents (alcohol or acetone), the raw glycopeptide is obtained in the form of a white to hazel powder. In the examples are reported some chemical analysis of this product, which is primarily characterized by the absence of uronic acids and the presence of only a trace of sulfur.

The subsequent purification treatments are intended especially to remove from the raw product, obtained according to the schemes hereinbefore illustrated, impurities of saline character and to allow the preparation of a material having a constant analytical composition.

Purification can be carried out essentially according to two schemes: either by dialysis (the impurities are easily dializable); or, by passing on exchange ion resins of acidic-type or mixed-type and in this case, instead, the purification is accomplished profiting by the strong ionic character of the polluting materials. In the examples, both of these purification processes are illustrated. The yield of the purification operation, whatever procedure is followed, vary from 65 to 75 percent by weight, and the product so obtained is in the form of a colorless, odorless and amorphous powder. The phosphorus and sulfur contents of the purified product are practically negligible. Also absent are the uronic acids, while, on the contrary, hexosamines are present in the range of 40-50 percent, amino acids in the range of 13-18 percent, hexoses in the range of 27-3 1 percent and neuraminic acid derivatives of 4-6 percent.

The product appears to be unitary to analytical investigations (electrophoresis, paper cromato'graph, etc.), and is precipitated by copper sulfate in an alkaline environment (biuret reagent) in quantitative yields.

The above analytical data permits that the products be classed among the glycopeptides, while, on the other hand, the presence of hexosamines not accompanied by uronic acids and the absence of sulfur-containing groupings of all kinds, confer a characteristic feature upon the polysaccharidic moiety of this substance.

Pharmacological tests on laboratory animals have proved that this glycopeptide, in a dose of 12.5, 25, or 50 mg./kg. inhibits the local edema by carragenine and serotonine, the diffusion of Blue Evans in the intradermal pomphi by serotonine, dextrane and bradiquinine, as well as the growth of cotton-pellets granuloma in the rat. At a dose of 100 mg./kg., the product being tested reduces the percentage of rats effected and the gravity of the pylorusligation ulcer according to Shay and the fast ulcer; moreover, it can promote the recovery of the functionality of inflamed limbs (arthritis by AgNO The following examples are to be construed as illustrative, but not limitative of the invention.

EXAMPLE 7 One hundred grams of raw glycoprotein obtained by the procedures described in examples 1 to 4 were suspended in 1 liter of water. The suspension was freed from the insoluble portion by centrifugation and the filtrate was treated with 100 ml. of Amberlite IR 120" resin (acidic form activated with 10 percent HCl; 26-50 U.S.S. mesh). After 30 minutes of this treatment, the resin was removed by filtration and washed with 100 ml. of distilled water; the wash liquors were combined with the filtrate. This solution, in turn, was treated with 100 ml. of Amberlite IRA 410 (basic form, activated with 5 percent NaOl-l: 20-50 U.S.S. mesh) for an additional 30 minutes. The resin was removed by filtration and washed with 150 ml. of distilled water; the wash liquors were combined with the filtrate and the pH value was brought to 6.2 by adding NaOH 2N. Five grams of sodium acetate were added, followed by dilution with an equal volume of acetone. The precipitate formed was collected by decantation, washed with acetone, dried under vacuum, and milled. Yield was 70 grams.

When operating on an industrial scale, it is more convenient to carry out the treatment with said ion exchange resins, by percolation in suitable columns, however, without any substantial modifications of the process.

The product obtained had the following analytical characteristics: P=0.04%; S=0.11%; Na=0.27%; acetyl groups 9.93%; N=5.74%; hexosamines 40%; uronic acids none; proteins (FOLlN) 14.5%; hexoses 31%; neuraminic acid derivatives 5.20%; pH 1 percent solution) 6.3; viscosity (on 1 percent solution), at 20 C.=l .9004.

COMPARATIVE EXAMPLE A The process of example 1 was repeated with one modification. Acidic hydrolysis byproducts formed by hydrolysis of the duodenum were not removed, rather acetone was added directly to the hydrolysis product.

The product, obtained in a yield of 150 grams, has the fol- TABLE 1 Tests Z inhibition at the doses (mg/kg.)

Carragenin edema 0 (+15) Shay ulcer 0 0 0 0 COMPARATIVE EXAMPLE B The process of example 1 was repeated with one modification. Acetone was added directly to the hydrolysis product and the resulting mixture was acidified with acetic acid. Then, the acidic hydrolysis byproducts were removed by decantatron.

Characteristics of the product which was obtained were:

Acetyl groups, 3.1% Viscosity 1 percent solution), 20 C., cps. 1.750

[-1 (1 percent solution) 5.7 ield of the product was about 130 grams. This procedure was substantially less advantageous than the process of example 1 in that the material obtained was found to be extremely impure and ineffective in the treatment of ulcers as demonstrated by the test data given in table 2 following.

While acetic and trichloroacetic acids have been shown above to be useful for acidifying the hydrolysis product, other acids can also be used. Typical of such acids are diluted hydrochloric and sulfuric acids.

What is claimed is:

1. Process for the preparation of a glycopeptide from the gastric mucosa or duodenum of swine, comprising:

a. hydrolyzing the animal organ in water at a temperature of from about 50 C. to about C. for from about 10 minutes to about 45 minutes at a pH of from about 1 to about 10;

b. acidifying the hydrolysis product of (a) whereby a precipitate and a liquid product are formed and separating said precipitate from the liquid product when hydrolysis of (a) is conducted at an alkaline pH, and centrifuging the hydrolysis product of (a) to separate a liquid product from solid matter when hydrolysis of (a) is conducted at an acidic pH;

c. subjecting the product of (b) to enzymatic proteolysis with papain or trypsin; and

d. diluting the product of (c) with a nonsolvent therefor,

' whereby a glycopeptide is precipitated therefrom.

2. Process of claim 1, wherein the product of (c) is dissolved in water, and the resulting aqueous solution is contacted with an acidic ion exchange resin before (d).

3. Process of claim 1, wherein the product of (d) is dissolved in water, the pH of the resulting solution is adjusted to a pH of 6 to 7, and the product is precipitated by addition of a nonsolvent therefor.

4. Process of claim 2, wherein contact with the acidic ion exchange resin is followed by contact with a basic ion exchange resin.

5. Process of claim 1, wherein the product is dialyzed at a pH from about 7 to about 8.

6. Process of claim 1, wherein the acidic byproducts of hydrolysis are precipitated by contacting the product of (a) with trichloroacetic acid.

7. Process of claim 1, wherein the pH of step (a) is from about 8.5 to about 9.5.

8. Process of claim 1, wherein the pH of step (a) is from about 1.5 to about 4.5.

9. Process of claim 13, wherein the nonsolvent is acetone or a lower alkanol. a

10. A glycopeptide extracted from the gastric mucosa or duodenum of swine having, by weight, a hexosamine content of from about 40 percent to 50 percent, a hexose content of from about 27 percent to 31 percent, an amino acid content of from about 13 percent to 18 percent, neuraminic acid derivatives of from about 4 percent to 6 percent, being substantially free from sulfur, phosphorus and uronic acids, and being prepared by the process of claim 1. 

2. Process of claim 1, wherein the product of (c) is dissolved in water, and the resulting aqueous solution is contacted with an acidic ion exchange resin before (d).
 3. Process of claim 1, wherein the product of (d) is dissolved in water, the pH of the resulting solution is adjusted to a pH of 6 to 7, and the product is precipitated by addition of a nonsolvent therefor.
 4. Process of claim 2, wherein contact with the acidic ion exchange resin is followed by contact with a basic ion exchange resin.
 5. Process of claim 1, wherein the product is dialyzed at a pH from about 7 to about
 8. 6. Process of claim 1, wherein the acidic byproducts of hydrolysis are precipitated by contacting the product of (a) with trichloroacetic acid.
 7. Process of claim 1, wherein the pH of step (a) is from about 8.5 to about 9.5.
 8. Process of claim 1, wherein the pH of step (a) is from about 1.5 to about 4.5.
 9. Process of claim 13, wherein the nonsolvent is acetone or a lower alkanol.
 10. A glycopeptide extracted from the gaStric mucosa or duodenum of swine having, by weight, a hexosamine content of from about 40 percent to 50 percent, a hexose content of from about 27 percent to 31 percent, an amino acid content of from about 13 percent to 18 percent, neuraminic acid derivatives of from about 4 percent to 6 percent, being substantially free from sulfur, phosphorus and uronic acids, and being prepared by the process of claim
 1. 